In Vitro Antimicrobial and Antibiofilm Properties and Bioaccessibility after Oral Digestion of Chemically Characterized Extracts Obtained from Cistus × incanus L., Scutellaria lateriflora L., and Their Combination

Periodontal diseases are oral inflammatory diseases ranging from gingivitis to chronic periodontitis. Porphyromonas gingivalis is one of the major pathogens responsible for severe and chronic periodontitis. Plant extracts with antimicrobial activity could be considered possible alternatives to chlorhexidine, an antiseptic substance used in oral hygiene thatcan cause bacteria resistance. Here, two commercial extracts obtained from Cistus × incanus L. and Scutellaria lateriflora L. were chemically characterized usingUltra-High-Performance Liquid Chromatography (UHPLC) coupled with a Q-Exactive Hybrid Quadrupole Orbitrap Mass Spectrometer. The extracts were studied for their bioaccessibility after simulated in vitro oral digestion, their antimicrobial activity against P. gingivalis, their protective effects against cellular invasion by P. gingivalis, and their antibiofilm activity. The extracts were found to contain very complex mixtures of polyphenols, which were quite stable after in vitro simulated oral digestion and demonstrated mild, dose-dependent inhibitory activity against P. gingivalis growth. This activity increased with the combination of the two extracts. Moreover, the combination of the extracts induced a reduction in P. gingivalis HaCaT invasiveness, and the reduction in biofilm came to around 80%. In conclusion, a combination of C. incanus and S. lateriflora showed promising effects useful in the treatment of gingivitis.

Cistus × incanus L. belongs to the family Cistaceae and is widespread along the Mediterranean coast of Europe. It was used as an effective anti-inflammatory and skin protective plant agent in Mediterranean folk medicines. Moreover, the use of C. incanus tea to rinse the mouth contributes to the degradation of biofilm, a well-known virulence factor, and the prevention of biofilm-induced diseases by decreasing the load of associated bacteria [19]. In addition to these properties, the extracts obtained from some Cistus species with compositions similar to C. incanus extracts are known for their antibacterial activity against oral cavity pathogens and have been suggested as alternative natural antibacterial and antibiofilm components against oral infections [20]. Scutellaria lateriflora L., also known as American skullcap, belongs to the family Lamiaceae and is one of most widely used nervine agents in North American and Western herbal medicine. Traditionally, it has been used to promote a healthy menstrual cycle and to treat hysteria, anxiety, insomnia, delirium tremens, epilepsy, withdrawal from barbiturates and tranquilizers, bronchitis, diarrhea, dysentery, jaundice, hepatitis, hypertension, thrombosis, and tumors [21]. Moreover, Scutellaria baicalensis shows synergistic antibacterial effects against oral bacterial biofilms in combination with chlorhexidine [22].
The increasing microbial resistance to chlorhexidine calls for adecrease in its use and the discovery of new combinations of plant extracts, which can act synergistically, with high antibacterial and antibiofilm properties. On the basis of the above information, the aim of this studywas to continue the researchon C. incanus and to evaluate the anti-gingivitis properties of S. lateriflora extracts, both alone and in combination with one another. This was done with the final aim of providing scientific evidence for the development of a new food supplement based on botanical extracts that is able to act at the oral-cavity level to prevent against periodontal diseases and improve the health of the oral cavity. Thus, two commercial extracts obtained from C. incanus and S. lateriflora were chemically characterized, and their oral bioaccessibility after in vitro simulated oral digestion was determined. Then, their in vitro antimicrobial activity against P. gingivalis, antibiofilm activity, and ability to enhance the barrier function of a gingival keratinocyte model system and exert a protective effect against invasion by P. gingivalis were evaluated.

2.2.
Chemical Characterization of C. incanus and S. lateriflora Extracts Using Reversed-Phase, Ultra-High-Performance Liquid Chromatography (RP-UHPLC) Coupled with a Q-Exactive Hybrid Quadrupole Orbitrap Mass Spectrometer Stock solutions were prepared for the C. incanus and S. lateriflora extracts by accurately weighing 200 mg of extract and diluting them with a solution of 50:50 v/v acidified water (0.1% v/v formic acid) and methanol to a concentration of 10 mg/mL. From the stock solutions, 1 mL was taken and filtered prior to analysis (0.45 µm and 0.20 µm Minisart RC 4 membrane filters). The analysis wasperformed on a Thermo Ultimate RS 3000 paired online with a Q-Exactive hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ionization probe (HESI II). For the RP-UHPLC analysis, a Kinetex ® EVOTM 150 mm × 2.1 mm, 2.6 µm (L × I.D, particle size, Phenomenex ® , Bologna, Italy) column was employed at a flow rate of 0.4 mL/min. The mobile phases consisted of (A) 0.1% CH 3 COOH in H 2 O and (B) ACN plus 0.1% CH 3 COOH. The analysis was performed in a gradient as follows: 0-10.0 min, 2-35% B; 10-12 min, 35-70% B; 12-13 min, 70-98% B; hold for 2 min; and return to initial conditions after 0.1 min. The column oven was set to 40 • C and 5 µL of the extracts were injected. An HRMS analysis was performed with Full MS (m/z 100-850) and data-dependent acquisition (dd-MS2 top; N = 5

In Vitro Bioaccessibility of C. incanus and S. lateriflora Extracts Using Simulated Oral Digestion Processes and RP-UHPLC-Photodiode Array Detector (PDA) Analysis
The current study aims to evaluate the possibility of a new food supplement for acting locally in the oral cavity against the pathogens associated with periodontal diseases; thus, the impact of the oral digestion process was verified on the chemical composition of C. incanus and S. lateriflora extracts following a protocol set by Minekus et al. with some modifications [24]. In brief, 5 g of each extract were dissolved in 3.5 mL of previously prepared, simulated salivary fluid (SSF) comprising an electrolyte solution containing (K + ), (Na + ), (Cl − ), (H 2 PO 4 − ), (HCO 3 − , CO 3 2− ), (Mg 2+ ), (NH 4 + ), and (Ca 2+ ). The same procedure was followed for the blank sample using 5 mL of water instead of the extracts. Then, 0.5 mL (1500 U/mL) of fresh α-amylase solution was added to both samples. In the end, water was added for the samples to reach a final volume of 10 mL, and the samples were incubated for 2 min at 37 • C. At the end of the oral digestion process, the samples were freeze dried and maintained at 4 • C prior to the RP-UHPLC-PDA analysis, which was performed on a Shimadzu Nexera LC30 (Shimadzu, Kyoto, Japan) with the same chromatographic conditions reported above; chromatograms were extracted at 280 and 330 nm.

Antimicrobial Activity of C. incanus and S. lateriflora Extracts against P. gingivalis
To evaluate the antimicrobial activity of C. incanus extract, S. lateriflora extract, their combinations in different ratios with the final concentrations of 60 mg/mL, and their carrier agents (i.e., maize maltodextrin and arabic gum), P. gingivalis (ATCC 33277), obtained from the LGC spa (ATCC distributor, Milan, Italy), was grown in a TSB-yeast extract medium supplemented with 0.05% cysteine hydrochloride, 0.02 µg/mL menadione, 5 µg/mL hemin, and 0.02% potassium nitrate in an anaerobic chamber (with 5% CO 2 ) at 37 • C. In brief, serial dilutions of the samples were prepared at volumes of 100 µL/well in 96-well plates. The final concentrations of each of these were in the range of 60 to 5 mg/mL. To each well 20 µL of P. gingivalis, bacterial cell suspension was added at a final concentration of 1 × 10 6 colony-forming units (CFU)/mL. Amoxicillin (10 µg/mL) was used as a positive control. After incubation in an anaerobic chamber at 37 • C for 24 h, bacterial growth was then analyzed using a microplate reader (Tecan, Männedorf, Swiss) at 595 nm. Each test was performed in triplicate. The rate of growth inhibition was determined using the following formula:  Human immortalized keratinocytes (HaCaT) were grown as monolayers in a standard culture medium, Dulbecco's Modified Eagle Medium (DMEM-10928_Gibco), and supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37 • C in a humidified atmosphere of 5% CO 2 and 95% air. The medium was replaced every 48 h. The trypsinization process for HaCaT cells was always performed at 70% confluence.

Cytotoxic Activity of C. incanus and S. lateriflora Extracts on HaCaT Cells
To assess the cytotoxic activity of the C. incanus and S. lateriflora extracts on HaCaT, alone and in combination, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. The MTT assay measures cellular metabolic activity as an indicator of vitality, proliferation, and cellular cytotoxicity [25]. Cells were cultured in DMEM and supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum at 37 • C with 5% CO 2 in a humid environment, as previously described. A density of 5 × 10 4 cells/well was seeded into 96-well plates and incubated for 24 h with (1) C. incanus extract at different concentrations ranging from 5 to 60 mg/mL, (2) S. lateriflora extract at different concentrations ranging from 5 to 60 mg/mL, or (3) their combinations in different ratios with a final concentration of 60 mg/mL. After 24 h of treatment, 100 µL of MTT solution (at a final concentration 0.5 mg/mL) was added to each well for 3 h at 37 • C. Then, the formazan crystals were solubilized by adding 100 µL of 100% DMSO to each well, and the viability rate was recorded at OD at 570 nm using a microplate reader (Tecan, Männedorf, Switzerland). Each test was performed in triplicate.

In Vitro Effects of C. incanus and S. lateriflora Extracts on Invasive Capacity of P. gingivalis Targeting HaCaT Cells
To investigate the effectiveness of the test extracts, alone and in combination, on the invasive capacity of P. gingivalis, an invasion assay was performed as described elsewhere [26]. HaCaT cells were seeded into 24-well plates (1 × 10 5 cells/well) and grown to~70-80% confluence. The day after the pre-treatment experimental scheme, the cell monolayers were starved for 2 h in a DMEM-10928_Gibco medium without antibiotics and were treated with the samples at different concentrations ranging from 15 to 60 mg/mL for 1 at 37 • C. After preincubation with the samples, cells were infected with 1.5 × 10 8 CFU/mL P. gingivalis. In a parallel co-treatment experimental scheme, bacteria and samples at the same concentrations reported above were incubated for 1 h and then used for the cell monolayer treatment. For both experimental schemes, after 4 h of infection, cells were washed with PBS three times and then incubated with gentamicin (Sigma-Aldrich, 100 µg/mL) to kill all extracellular bacteria. After 2 h in the presence of gentamicin, cells were lysed with 0.1% Triton-X solution to evaluate the amountof intracellular bacteria. Serial dilutions of the cell lysates were made in PBS, plated on TB agar, and incubated at 37 • C overnight. Then, the CFU/mL was counted relative to the bacteria that invaded the cell monolayer after incubation for 24 h at 37 • C.

Effects of C. incanus and S. lateriflora Extracts on Pre-Formed Biofilm Mass Reduction
The ability for C. incanus and S. lateriflora extracts to degrade pre-formed biofilm was evaluated using acrystal violet (CV) assay [27]. Briefly, a bacterial inoculum was prepared at a density of 1 × 10 8 CFU/mL in TSB supplemented with 1% glucose. A volume of 100 µL of bacterial suspension was transferred to each well of a 96-well plate and incubated at 37 • C for 24 h under static conditions to allow biofilm formation. After incubation, non-adherent cells were removed through PBS washes, and C. incanus and S. lateriflora extracts at different concentrations ranging from 5 to 55 mg/mL were added to the mature biofilm. The untreated and EDTA-treated biofilms constituted negative and positive controls, respectively. After treatment, the growth medium was removed, and the biofilm was gently washed with PBS. The biofilm biomass was quantified by adding 100 µL of 0.1% CV to each well for 30 min at room temperature under shaken conditions. Excess CV was removed, washed with PBS, and then solubilized with 98% ethanol for 40 min at room temperature under shaking conditions. Absorbance values recorded at 570 nm using a microplate reader (Tecan, Männedorf, Swiss) and were proportional to the biofilm mass present, and the results were expressed by calculating the percentage of reduction of the biofilm mass compared to the control samples.

Statistical Analysis
Data are reported as mean ± standard deviation (SD). The bacterial growth percentage and the cell invasive capacity were compared with the control sample at each examination point using an independent samples t-test, setting the level of significance at p < 0.05. Moreover, a statistical comparison among groups was conducted with multiple t-tests for multiple comparison using the Holm-Sidak method to analyze the bacterial growth inhibition percentage or the biofilm mass reduction percentage induced by the plant extract combinations to determine significance, which was set to p < 0.05. For each concentration used in both the antimicrobial and antibiofilm activities, a biological replicate was obtained and averaged. The statistical analyses were performed using GraphPad Prism, version 8 (San Diego, CA, USA).

Metabolic Profile of C. incanus and S. lateriflora Extracts
The first step was the chemical characterization of the commercial hydroalcoholic C. incanus and S. lateriflora extracts using RP-UHPLC coupled with a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer. Through a comparison with in silico MS/MS spectra, accurate mass, and molecular formula, 101 and 117 compounds were tentatively annotated in C. incanus and S. lateriflora extracts, respectively, with confidence MSI lvl.2 [28] as reported in the Supplementary Tables. The base peak chromatograms are reported in Figure 1.

Bioaccessibility of C. incanus and S. lateriflora Extracts after In Vitro Simulated Oral Digestion
To evaluate the influence of the in vitro simulated oral digestion process on the compounds most represented in C. incanus and S. lateriflora extracts, they were analyzed using RP-UHPLC-PDA before and after oral digestion. A slight shift in retention times

Bioaccessibility of C. incanus and S. lateriflora Extracts after In Vitro Simulated Oral Digestion
To evaluate the influence of the in vitro simulated oral digestion process on the compounds most represented in C. incanus and S. lateriflora extracts, they were analyzed using RP-UHPLC-PDA before and after oral digestion. A slight shift in retention times was observed for orally digested samples, probably due to a matrix effect reduction following in vitro simulated oral digestion. UV traces of C. incanus and S. lateriflora extracts before and after in vitro simulated oral digestion are reported in Figures 2 and 3, respectively.   Table 1 shows the mean peak area reduction percentages, ranging from 6.4 to 11.6%, after oral digestion of C. incanus extract, with the exception of kaempferol 3-(3 -p-coumaoroylhexoside), which was degraded by 46% when compared with the peak area recorded before digestion, revealing a moderate degradation process. Table 2 shows the mean peak area reduction percentage after oral digestion of S. lateriflora extract main peaks, which was found to be lower than 10%, revealing a modest degradation process.    Table 1 shows the mean peak area reduction percentages, ranging from 6.4 to 11.6%, after oral digestion of C. incanus extract, with the exception of kaempferol 3-(3″-p-coumaoroylhexoside), which was degraded by 46% when compared with the  3.3. Antibacterial Activity of C. incanus and S. lateriflora Extracts against P. gingivalis C. incanus and S. lateriflora extracts were tested for their antibacterial activity against P. gingivalis. The results showed mild dose-dependent bacterial growth inhibitory activity, which did not allow for the determination of the minimum inhibitory concentration (MIC) values. In fact, at the highest concentration (60 mg/mL) at the end of the treatment, the percentage of P. gingivalis growth was found to be reduced to 55% and 53% for C. incanus and S. lateriflora, respectively (Figure 4), in comparison to the control sample, in which P. gingivalis was grown without the extracts. In particular, compared to the control sample, the statistically significant differences in the inhibition of microbial growth by C. incanus extract and S. lateriflora extract treatments were recorded starting from the concentration of 40 mg/mL (p = 0.0025) and 20 mg/mL (p = 0.0198), respectively ( Figure 4).
As expected, neither maltodextrin nor arabic gum exerted inhibitory effects on the bacterial growth.
Considering the low recorded antibacterial activity, the extracts used alone (at the concentrations of 5, 15, 20, and 30 mg/mL) and their combinations in different ratios (i.e., 1:1, 1:2, 1:3, etc.) with the final concentrations of 60 mg/mL were subjected to the same test. The results, reported in Figure 5, are expressed as percentage of bacterial growth inhibition. As regards the antimicrobial activities of C. incanus and S. lateriflora used alone, the results of the statistical analysis have already been reported in Figure 4. As regards the antimicrobial activities of C. incanus and S. lateriflora combinations, a comparison between the bacterial growth percentage recorded following the treatment with C. incanus and S. lateriflora combinations and the bacterial growth percentage recorded in their absence (control sample) shows a significance in every combination (p < 0.05), including those combinations in which C. incanus used alone at concentrations lower than 40 mg/mL was not found to be effective. 3.3. Antibacterial Activity of C. incanus and S. lateriflora Extracts against P. gingivalis C. incanus and S. lateriflora extracts were tested for their antibacterial activity against P. gingivalis. The results showed mild dose-dependent bacterial growth inhibitory activity, which did not allow for the determination of the minimum inhibitory concentration (MIC) values. In fact, at the highest concentration (60 mg/mL) at the end of the treatment, the percentage of P. gingivalis growth was found to be reduced to 55% and 53% for C. incanus and S. lateriflora, respectively (Figure 4), in comparison to the control sample, in which P. gingivalis was grown without the extracts. In particular, compared to the control sample, the statistically significant differences in the inhibition of microbial growth by C. incanus extract and S. lateriflora extract treatments were recorded starting from the concentration of 40 mg/mL (p = 0.0025) and 20 mg/mL (p = 0.0198), respectively (Figure 4).   As expected, neither maltodextrin nor arabic gum exerted inhibitory effects on the bacterial growth.
Considering the low recorded antibacterial activity, the extracts used alone (at the concentrations of 5, 15, 20, and 30 mg/mL) and their combinations in different ratios (i.e., 1:1, 1:2, 1:3, etc.) with the final concentrations of 60 mg/mL were subjected to the same test. The results, reported in Figure 5, are expressed as percentage of bacterial growth inhibition. As regards the antimicrobial activities of C. incanus and S. lateriflora used alone, the results of the statistical analysis have already been reported in Figure 4. As regards the antimicrobial activities of C. incanus and S. lateriflora combinations, a comparison between the bacterial growth percentage recorded following the treatment with C. incanus and S. lateriflora combinations and the bacterial growth percentage recorded in their absence (control sample) shows a significance in every combination (p < 0.05), including those combinations in which C. incanus used alone at concentrations lower than 40 mg/mL was not found to be effective. Interestingly, the results show that, for the combinations of C. incanus and S. lateriflora at concentration ratios of 1:3, 1:2, and 1:1, the percentages of bacterial growth inhibition are greater than the sum of the percentages of bacterial growth inhibition recorded for the individual C. incanus and S. lateriflora extracts at the same concentrations. More specifically, we compared the sums of the bacterial growth inhibitory activities induced by the treatments with C. incanus and S. lateriflora extracts used alone with the bacterial growth inhibitory activities induced by the combinations of these extracts using a multiple t-test analysis. As regards the combinations of C. incanus and S. lateriflora (at concentration ratios of 1:3, 1:2, and 1:1), the differences between the sum of the activities of the individual extracts and the activities of these combinations was found to be statistically significant (p < 0.001, p < 0.05, and p < 0.0001, respectively), with the combinations of extracts showing higher activities. In contrast, in the other cases, the sum of the activities of the individual extracts was greater when compared to the effects shown by the combinations of the two extracts ( Figure 6). Interestingly, the results show that, for the combinations of C. incanus and S. lateriflora at concentration ratios of 1:3, 1:2, and 1:1, the percentages of bacterial growth inhibition are greater than the sum of the percentages of bacterial growth inhibition recorded for the individual C. incanus and S. lateriflora extracts at the same concentrations. More specifically, we compared the sums of the bacterial growth inhibitory activities induced by the treatments with C. incanus and S. lateriflora extracts used alone with the bacterial growth inhibitory activities induced by the combinations of these extracts using a multiple t-test analysis. As regards the combinations of C. incanus and S. lateriflora (at concentration ratios of 1:3, 1:2, and 1:1), the differences between the sum of the activities of the individual extracts and the activities of these combinations was found to be statistically significant (p < 0.001, p < 0.05, and p < 0.0001, respectively), with the combinations of extracts showing higher activities.
In contrast, in the other cases, the sum of the activities of the individual extracts was greater when compared to the effects shown by the combinations of the two extracts ( Figure 6).

Modulating Effects of C. incanus and S. lateriflora Extracts and Their Combinations on P. gingivalis Cell Invasive Capacity
To evaluate the activity of C. incanus and S. lateriflora extracts and their combinations on reducing the invasiveness of P. gingivalis in a HaCaT model system, HaCaT cells were treated with non-cytotoxic concentrations of C. incanus and S. lateriflora extracts (ranging from 10 to 60 mg/mL) before infection with P. gingivalis. The same was performed for the extract combinations at different ratios (i.e., 1:1, 1:2, 1:3, etc.) with final concentrations of 60 mg/mL (pre-treatment experimental condition). In addition, the co-treatment of Ha-CaT cells with P. gingivalis and the extracts used alone or in combination was also performed (co-treatment experimental condition). The results show that only the combination of C. incanus and S. lateriflora at the highest tested concentration (60 mg/mL), in the ratio 1:1, reduced the invasiveness of P. gingivalis, reflected as 6.4 × 10 5 ± 1.1 × 10 4 CFU/mL, compared to the control of 6.4 × 10 7 ± 2.0 × 10 5 CFU/mL (p = 0.0006). The other samples showed no modulation of the bacterial invasive capacity.

Effects of C. incanus and S. lateriflora Extracts and Their Combinations on the Degradation of Pre-Formed P. gingivalis Biofilm
Biofilm formation is one of the main virulence mechanisms of P. gingivalis, contributing to an increase in the gingival tissue degradation process. The activity of C. incanus and S. lateriflora extracts and their combinations was evaluated on pre-formed biofilm. The biofilm biomass was quantified using CV in response to the treatments with the extracts used alone (at concentrations ranging from 5 to 55 mg/mL) and their combinations in different ratios (i.e., 1:1, 1:2, 1:3, etc.) with the final concentrations of 60 mg/mL, and then compared to measurements of the untreated mature biofilm. After 20 h of incubation, the samples induced a reduction in biofilm mass ranging from 11 to 56% and from 15 to 68% for the individual C. incanus and S. lateriflora, respectively. The combinations of the extracts in different ratios induced a biofilm mass reduction of about 80% (Figure 7). The statistical analysis shows that, by comparing the mass of biofilm produced by P. gingivalis following the treatment with C. incanus and S. lateriflora extracts used alone, in combination, and in their absence (control sample), we observed a statistically significant difference startingat the first tested concentration (5 mg/mL) (p < 0.05). Results are expressed as mean ± SD from three biological replicated (n = 3). *: p < 0.05, ***: p < 0.001, ****: p < 0.0001.

Modulating Effects of C. incanus and S. lateriflora Extracts and Their Combinations on P. gingivalis Cell Invasive Capacity
To evaluate the activity of C. incanus and S. lateriflora extracts and their combinations on reducing the invasiveness of P. gingivalis in a HaCaT model system, HaCaT cells were treated with non-cytotoxic concentrations of C. incanus and S. lateriflora extracts (ranging from 10 to 60 mg/mL) before infection with P. gingivalis. The same was performed for the extract combinations at different ratios (i.e., 1:1, 1:2, 1:3, etc.) with final concentrations of 60 mg/mL (pre-treatment experimental condition). In addition, the co-treatment of HaCaT cells with P. gingivalis and the extracts used alone or in combination was also performed (co-treatment experimental condition). The results show that only the combination of C. incanus and S. lateriflora at the highest tested concentration (60 mg/mL), in the ratio 1:1, reduced the invasiveness of P. gingivalis, reflected as 6.4 × 10 5 ± 1.1 × 10 4 CFU/mL, compared to the control of 6.4 × 10 7 ± 2.0 × 10 5 CFU/mL (p = 0.0006). The other samples showed no modulation of the bacterial invasive capacity.

Effects of C. incanus and S. lateriflora Extracts and Their Combinations on the Degradation of Pre-Formed P. gingivalis Biofilm
Biofilm formation is one of the main virulence mechanisms of P. gingivalis, contributing to an increase in the gingival tissue degradation process. The activity of C. incanus and S. lateriflora extracts and their combinations was evaluated on pre-formed biofilm. The biofilm biomass was quantified using CV in response to the treatments with the extracts used alone (at concentrations ranging from 5 to 55 mg/mL) and their combinations in different ratios (i.e., 1:1, 1:2, 1:3, etc.) with the final concentrations of 60 mg/mL, and then compared to measurements of the untreated mature biofilm. After 20 h of incubation, the samples induced a reduction in biofilm mass ranging from 11 to 56% and from 15 to 68% for the individual C. incanus and S. lateriflora, respectively. The combinations of the extracts in different ratios induced a biofilm mass reduction of about 80% (Figure 7). The statistical analysis shows that, by comparing the mass of biofilm produced by P. gingivalis following the treatment with C. incanus and S. lateriflora extracts used alone, in combination, and in their absence (control sample), we observed a statistically significant difference startingat the first tested concentration (5 mg/mL) (p < 0.05). Foods 2023, 12, x FOR PEER REVIEW 12 of 16 Figure 7. A comparison between the mass of biofilm produced by P. gingivalis following treatment with C. incanus and S. lateriflora extracts alone, in combination, and in their absence (control) showed a significance starting at the first concentration tested (5 mg/mL).
As regards the percentage of biofilm mass reduction inhibition, the sums of the activities exerted by C. incanus and S. lateriflora extracts used alone and those of the extract combinations were compared through a multiple t-test analysis. The results showed that the sum of the activities exerted by C. incanus and S. lateriflora used alone were greater compared to the effects exerted by the combinations of the two extracts ( Figure 8). In this case, no significant difference was demonstrated (p < 0.05).

Discussion
P. gingivalis is one of the major pathogens responsible for severe and chronic manifestations of periodontal disease, producing a number of virulence factors that cause direct and indirect destruction to periodontal tissues through modulation of the host inflammatory response [29]. Although human gingival epithelium prevents intrusion by periodontal bacteria, P. gingivalis is able to invade gingival epithelial cells. Without any doubt, primary prevention (including the use of toothbrushes, dental floss, water picks, toothpicks, small interproximal brushes, rubber gum stimulators, and mouthwash with antimicrobial and antibiofilm activities) is not only the most effective but also the Figure 7. A comparison between the mass of biofilm produced by P. gingivalis following treatment with C. incanus and S. lateriflora extracts alone, in combination, and in their absence (control) showed a significance starting at the first concentration tested (5 mg/mL).
As regards the percentage of biofilm mass reduction inhibition, the sums of the activities exerted by C. incanus and S. lateriflora extracts used alone and those of the extract combinations were compared through a multiple t-test analysis. The results showed that the sum of the activities exerted by C. incanus and S. lateriflora used alone were greater compared to the effects exerted by the combinations of the two extracts ( Figure 8). In this case, no significant difference was demonstrated (p < 0.05). As regards the percentage of biofilm mass reduction inhibition, the sums of the activities exerted by C. incanus and S. lateriflora extracts used alone and those of the extract combinations were compared through a multiple t-test analysis. The results showed that the sum of the activities exerted by C. incanus and S. lateriflora used alone were greater compared to the effects exerted by the combinations of the two extracts ( Figure 8). In this case, no significant difference was demonstrated (p < 0.05).

Discussion
P. gingivalis is one of the major pathogens responsible for severe and chronic manifestations of periodontal disease, producing a number of virulence factors that cause direct and indirect destruction to periodontal tissues through modulation of the host inflammatory response [29]. Although human gingival epithelium prevents intrusion by periodontal bacteria, P. gingivalis is able to invade gingival epithelial cells. Without any doubt, primary prevention (including the use of toothbrushes, dental floss, water picks, toothpicks, small interproximal brushes, rubber gum stimulators, and mouthwash with antimicrobial and antibiofilm activities) is not only the most effective but also the

Discussion
P. gingivalis is one of the major pathogens responsible for severe and chronic manifestations of periodontal disease, producing a number of virulence factors that cause direct and indirect destruction to periodontal tissues through modulation of the host inflammatory response [29]. Although human gingival epithelium prevents intrusion by periodontal bacteria, P. gingivalis is able to invade gingival epithelial cells. Without any doubt, primary prevention (including the use of toothbrushes, dental floss, water picks, toothpicks, small interproximal brushes, rubber gum stimulators, and mouthwash with antimicrobial and antibiofilm activities) is not only the most effective but also the cheapest way of coping with periodontitis and its complications [30]. Considering microbial resistance to the antibacterial agents, scientific research draws its attention towards the assessment of plant extracts (rich in bioactive phytochemicals, i.e., flavonoids, alkaloids, tannins, and terpenoids) with antimicrobial activity that may counteract emerging microbial resistance, while exhibiting antimicrobial properties against P. gingivalis [31]. In this study, we demonstrate that acombination of two commercial extracts obtained from C. incanus and S. lateriflora, which consists of a complex mixture of bioactive compounds that is stable after in vitro simulated oral digestion, decreases the in vitro growth of P. gingivalis and enhances the barrier function of a gingival keratinocyte model system, exerting both protective effects against invasion by P. gingivalis and antibiofilm activity. In more detail, as far as the phytochemical profiles of C. incanus and S. lateriflora extracts are concerned, more than one hundred compounds were identified for each extract. The results obtained support previous reports on the phytochemical compositions of C. incanus and S. lateriflora extracts, although to date no study has determined the metabolic profile of these extracts in such detail [21,[32][33][34][35][36][37][38][39]. Moreover, the bioaccessibility of C. incanus and S. lateriflora polyphenolic compounds, to which their antibacterial and antibiofilm activities against periodontal pathogens are ascribed, has a strong impact on their ability to exert their biological activities in the oral cavity and was assessed. To the best of our knowledge, no earlier investigation has been published indicating the bioaccessibility of C. incanus and S. lateriflora polyphenols after oral digestion, and we are reporting for the first time that the concentrations of the most represented polyphenols occurring in C. incanus and S. lateriflora extracts are stable under oral digestion conditions.
As far as the antibacterial activities of C. incanus and S. lateriflora extracts against P. gingivalis are concerned, our results show a mild inhibitory effecton bacterial growth for both extracts in a dose-dependent manner. Interestingly, the combinations of the extracts exerted a greater inhibition of bacterial growth, especially when S. lateriflora is present in the culture medium in higher concentrations than those of C. incanus. To the best of our knowledge, the antimicrobial activities of C. incanus and S. lateriflora extracts alone and in combination against P. gingivalis have never been studied. These results could be considered in agreement with data from the existing literature that demonstrates the antimicrobial properties of C. incanus against Gram-positive pathogens (i.e., S. aureus and S. epidermidis) [40] and Streptococcus mutans colonization on enamel samples exposed to oral fluids [34]. Moreover, S. lateriflora root extract showed antibacterial effects against Bacillus subtilis (NCIMB 3610) and E. coli (NCIMB 8879) with a minimum inhibition concentration of 5 mg/mL and 25 mg/mL, respectively [41]. Moreover, the combination of C. incanus and S. lateriflora yielded a slight reduction in the cellular invasiveness of P. gingivalis at the highest tested concentration in pre-treatment assay conditions. To the best of our knowledge, no earlier investigation has been published on the reduction in the cellular invasiveness of P. gingivalis in the presence of C. incanus and S. lateriflora extracts. The invasion of host cells is the first step for bacteria to establish pathogenic reservoirs and evade host defense mechanisms [42]. Furthermore, while the human gingival epithelium prevents intrusion by periodontal bacteria, P. gingivalis is able to invade gingival epithelial cells (primarily through the action of P. gingivalis proteases), breaking the oral epithelial barrier and spreading into periodontal tissues [42]. Thus, although the reduction in invasiveness recorded is moderate, it could contribute to a reduction in infection and periodontal tissue injury. Finally, as regards the capability of C. incanus and S. lateriflora to modulate mature P. gingivalis biofilm, known as subgingival plaque, the results show that the combination of these extracts is capable of the almost total degradation of biofilm. This result is all the more important considering that bacteria within a biofilm have shown 10-1000 times more antibiotic resistance than planktonic bacteria [43]. Oral biofilm protects bacteria from counteraction by the host's immune system and antibacterial agents in vivo. Furthermore, cells embedded in biofilm are more resistant to antibiotic treatment as the virulence factors produced by P. gingivalis are contained within the biofilm, such as fimbriae, hemagglutinins, and proteinases [44]. The results obtained support previous investigations showing the antibiofilm activity of the extracts obtained from different species of the Cistus genus (i.e., C. creticus L., C. monspeliensis L., and C. laurifolius L.) and Scutellaria genus (i.e., S. baicalensis) against biofilm formation of Gram-positive bacteria (S. aureus, Bacillus subtilis, and S. mutans) and Gram-negative bacteria (E. coli, S. enterica, P. aeruginosa, and K. pneumonia) [20,22,45,46].
In conclusion, the combined effects of C. incanus and S. lateriflora in the inhibition of the growth of P. gingivalis and its invasiveness as well asthe reduction of pre-formed biofilm mass may open considerations of their use in the treatment of gingivitis and as adjunctive therapeutic agents to periodontitis. Further in vitro studies reflecting insights into the mechanism of action and clinical trials assessing the efficacy of these extracts in human subjects are currently in progress.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods12091826/s1, Table S1. Identified compounds in C. incanus extract according to molecular formula, m/z, and the retention time (RT); Table S2. Identified compounds in S. lateriflora extract according to molecular formula, m/z, and the retention time (RT).